Osteosarcoma tumors maintain intertumoral heterogeneity, even while adapting to environmental pressures that drive clonal selection

Osteosarcoma exhibits marked patient-to-patient heterogeneity, but little is known about heterogeneity within individual tumors. This study focuses on the roles that phenotypic plasticity and clonal selection play as tumors adapt to primary and metastatic microenvironments. We show that osteosarcomas have a high degree of transcriptional heterogeneity, like osteoblasts, that is retained even after prolonged cell culture or adaptation to changing microenvironments. We find that both cell lines and PDXs grown in cell culture or as flank tumors adopt markedly different transcriptional profiles when grown as primary bone tumors or metastatic lung lesions. By combining lineage tracing with single-cell transcriptomics, we find that very little clonal selection occurs when tumors grow in the tibia, but significant expansion of select clones occurs when grown as experimental metastases in the lung. Interestingly, the selective pressures that drive clonal expansion do not cause narrowing of transcriptional phenotypes. By comparing the phenotypes from transcriptional clusters in orthotopic/metastatic tumor pairs, we identify a transcriptional signature that is shared among clusters that become enriched during lung colonization. This includes responses to IFNG, TNF, PDGF, previously unidentified IL1B, and a shift away from genes associated with glycolysis and toward those associated with oxidative metabolism. The metastatic microenvironment enriches for phenotypically diverse clones that each display metabolic properties that engender fitness within the metastatic microenvironment. Together, these data suggest that an underlying program, possibly a developmental program retained from the tissue of origin, maintains phenotypic heterogeneity, even during adaptation to changing microenvironmental conditions.